Na+-driven, secondary-active transporters are transmembrane proteins that concentrate ions or organic substrates on one side of the membrane by coupling substrate transport to the transmembrane electrochemical gradient for Na+. The aim of this proposal is to continue research towards the understanding of the fundamental principles by which these transporters work, focusing on the plasma membrane transporters for the neurotransmitter glutamate (excitatory amino acid transporters, EAATs). Although progress has been made towards this goal by the recent development of a structural model of glutamate transporters based on the x-ray structure of the bacterial aspartate transporter GltPh, important questions about the actual transport mechanism, the coupling of substrate transport to cation flux, and the function and regulation of various glutamate transporter subtypes remain unresolved. These questions will be addressed by undertaking research towards three specific aims. Aim 1 will address two gaps in knowledge that currently prevent us from understanding the overall transport mechanism by a) determining the sequence of individual events associated with Na+/glutamate movement across the membrane, and b) testing the hypothesis that K+ outward movement is a voltage-dependent, multi-step process, occurring through intermediate(s) on the translocation pathway. Aim 2 will identify structural elements of mammalian glutamate transporters contributing to their interaction with cations. Two hypotheses will separately be tested, both related to different aspects of the mechanism of cation interaction with EAATs: a) Only one of the two cation binding sites seen in GltPh corresponds to a Na+ binding site of mammalian EAATs;and b) cation binding site(s) exist on mammalian EAATs that are not seen in the GltPh structure. Aim 3 will comparatively analyze the functional properties of the neuronal glutamate transporter subtypes EAAT3, EAAT4 and EAAT5 and establish the role of the C-terminus in the regulation of EAAT5 activity. The hypothesis to be tested is that EAAT5 is a slowly-gated anion channel with little transport activity, in contrast to EAAT3 and EAAT4, which both transport glutamate. To approach these three aims, wild-type glutamate transporters and transporters with specific mutations to potential cation binding sites predicted from empirical valence mapping will be expressed in HEK293 cells, followed by functional analysis using uptake assays and transport current recording with <100 ms time resolution, allowing us to follow the dynamics of transport in real time. Understanding the molecular mechanism and the dynamics of glutamate transport and its regulation is important because these transporters not only contribute to controlling the time course of the excitatory neurotransmitter in the synaptic cleft, thus indirectly modulating glutamatergic transmission, but also directly regulate cell excitability through their anion channel function. PUBLIC HEALTH RELEVANCE: This proposal examines how neuronal glutamate transporters work. These transporters contribute to the regulation of neurotransmission by removing synaptically-released glutamate from the extracellular space, and by controlling neuronal excitability in the retina through their anion channel function. Abnormally high transport activity is implicated in Schizophrenia, whereas down-regulated transport activity is associated with neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS), Huntington's disease, Alzheimer's disease, and brain insults. Understanding the mechanism(s) by which glutamate is transported across the membrane, how the cations that drive uptake interact with the transporters, how the labor is divided between the five known members of the glutamate transporter family, and how transport is regulated will facilitate our comprehension of the contributions of glutamate transporters to CNS disorders and may lead to new approaches to treat them.